Mechanistic studies on Zymogen-activator and adhesion proteins (ZAAPs) as thrombolytic drugs and bacterial virulence factors

Streptokinase (SK), expressed by Lancefield Group A, C and G β-haemolytic Streptococci and Staphylocoagulase (SCG), expressed by S. aureus, are bacterial virulence factors which belong to a family of proteins known as Zymogen-activator and adhesion proteins (ZAAPs). SK and SCG are responsible for the non-proteolytic activation of plasminogen and prothrombin, respectively. Understanding of SK activity is exclusively based on the Group C (GCS) S. equisimilis H46a SK, a ‘clot buster’ or thrombolytic used in the treatment of Myocardial Infarction (MI), which exhibits no fibrin specificity. SK is the most used thrombolytic worldwide. Here, detailed kinetic studies in purified assay systems explored the mechanistic variation between a recombinant H46a SK (rSK H46a) and a Group A Streptococcal SK (M1GAS), most typically isolated in invasive human infection. This work demonstrates a fibrin specific mechanism for M1GAS SK and proposes a kinetic model for M1GAS SK plasminogen activation, to compliment the “Trigger and Bullet” hypothesis for H46a SK by Bock and colleagues. This work has relevance to the use of SK variants, with enhanced fibrin specificity, for improvement of thrombolytic therapies. Cardiovascular diseases such as myocardial infaraction and ischaemic stroke are significant casues of mortality, particularly in the developing world. Access to Alteplase, an expensive recombinant tPA and the only licensed treatment for stroke, is limited and there is interest in the use of SK for this purpose. Furthermore, microbial resistance is an increasing health burden, as demonstrated by programs such as the Longitude prize. Exploring the mechanisms of bacterial virulence factors at the molecular level such as this could provide rationale for the development of much-needed new antimicrobial technologies.  Open Acces

Assessing the risks of haemolysis as an adverse reaction following the transfusion of ABO incompatible plasma-containing components - a scoping review

Background: The limited supply of universal plasma has resulted in transfusion of ABO incompatible plasma to patients. As the need to implement whole blood transfusion in pre-hospitals setting rises, the lowest cut-off for anti-A/anti-B that does not cause haemolysis remains unknown. In this first scoping review, we aimed to determine the lowest ABO titre and volume reported in the literature to cause haemolysis from ABO incompatible plasma transfusions (plasma, platelets, cryoprecipitate, and whole blood). Methods: We searched several databases from inception to April 2022, including all study types. Three independent reviewers extracted and reviewed the data. Primary outcome was the anti-A and anti-B titre (measured by IgM or IgG) that resulted in measurable haemolysis following ABO incompatible plasma transfusion. Results: We identified 5681 citations, of which 49 studies were eligible, reporting a total of 62 cases (34 adults, 14 children and 14 did not specify age). The methods for antibody measurement and antibody type (IgG or IgM) varied significantly between studies. Component volumes were poorly reported. The most common component responsible for the haemolysis was apheresis platelets followed by pooled platelets and whole blood. Most haemolytic cases reported were due to anti-A. The lowest anti-A titre reported to cause haemolysis (children and adults) was 32 (IgG), while for anti-B it was 512 (IgG and IgM) for adults, 16,384 for paediatrics (IgG and IgM) and 128 (IgM) in cases where the age was not specified. The lowest reported volume associated with haemolysis were 100 ml (adults) and 15 ml (children). Of the 62 15 (24%) died. Conclusion: The lowest titre reported to cause haemolysis was an anti-A of 32. ABO mismatch plasma transfusion may be associated with significant mortality. There is a need to agree/standardise methods for ABO titration measurement internationally for plasma components and agree the lowest anti-A/anti-B titre for transfusing ABO mismatched plasma

Scheme outlining the pathway of activation of Pgn by rSK-M1GAS and stimulation by Fgn.

<p>The scheme follows a trigger and bullet mechanism where an initial activator complex of SK•Pgn (BG) is replaced by SK•Pm (BE) as Pm (E) is generated due to the higher affinity binding of E to SK. Fgn (F) associates weakly with Pgn (G), while formation of active Michaelis complexes, GFBG and GFBE have improved dissociation constants. An improved rate of Pgn activation is achieved by GFBE relative to GFBG due to lower K_M, while the k_cat for formation of Pm is unchanged. Derivation of the constants shown is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.t002" target="_blank">Table 2</a>.</p

Activity Regulation by Fibrinogen and Fibrin of Streptokinase from Streptococcus Pyogenes

<div><p>Streptokinase is a virulence factor of streptococci and acts as a plasminogen activator to generate the serine protease plasmin which promotes bacterial metastasis. Streptokinase isolated from group C streptococci has been used therapeutically as a thrombolytic agent for many years and its mechanism of action has been extensively studied. However, group A streptococci are associated with invasive and potentially fatal infections, but less detail is available on the mechanism of action of streptokinase from these bacteria. We have expressed recombinant streptokinase from a group C strain to investigate the therapeutic molecule (here termed rSK-H46A) and a molecule isolated from a cluster 2a strain from group A (rSK-M1GAS) which is known to produce the fibrinogen binding, M1 protein, and is associated with life-threatening disease. Detailed enzyme kinetic models have been prepared which show how fibrinogen-streptokinase-plasminogen complexes regulate plasmin generation, and also the effect of fibrin interactions. As is the case with rSK-H46A our data with rSK-M1GAS support a “trigger and bullet” mechanism requiring the initial formation of SK•plasminogen complexes which are replaced by more active SK•plasmin as plasmin becomes available. This model includes the important fibrinogen interactions that stimulate plasmin generation. In a fibrin matrix rSK-M1GAS has a 24 fold higher specific activity than the fibrin-specific thrombolytic agent, tissue plasminogen activator, and 15 fold higher specific activity than rSK-H46A. However, in vivo fibrin specificity would be undermined by fibrinogen stimulation. Given the observed importance of M1 surface receptors or released M1 protein to virulence of cluster 2a strain streptococci, studies on streptokinase activity regulation by fibrin and fibrinogen may provide additional routes to addressing bacterial invasion and infectious diseases.</p></div

Inhibition of Pgn activation in a Fgn or fibrin environment by tranexamic acid (TA).

<p>Data are presented as the % activity remaining relative to activation with no TA where rSK-M1GAS (red symbols) or rSK-H46A (black symbols) is activator. Open symbols and dashed lines are for data in the presence of Fgn and solid symbols and lines are in fibrin. Curve fitting to a 4 parameter model suggests a significant difference between IC₅₀for rSK-M1GAS in the presence of Fgn (14.5 μM) and fibrin (133 μM). Inhibition of Pgn activation by rSK-H46A was inhibited at higher TA and there was no significant difference with Fgn or fibrin.</p

Fibrin clot lysis by tPA, rSK-M1GAS and rSK-H46A over a range of Pgn concentrations.

<p>Fibrin clots were prepared using 3 mg/ml Fgn and incorporating Pgn from 0–1.6 μM. Rate of clot lysis was estimated from time to 50% clot lysis, as 1000x 1/time to 50% lysis in seconds. To get similar rates, activator concentrations used were 0.6 M tPA (blue circles), 0.3 nM rSK-H46A (black squares) and 0.02 nM rSK-GASM1 (red triangles). Detailed results from fitting to the Michaelis-Menten equation are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.t003" target="_blank">Table 3</a>.</p

The effect of known stimulators on Pgn activation by tPA, rSK-M1GAS and rSK-H46A.

<p>Bars show the degree of stimulation (rate with stimulator/rate without stimulator) for fixed concentrations of Pgn and tPA (blue), rSK-M1GAS (red) and rSK-H46A (black), using both glu- or lys-Pgn as substrate. Abbreviations are Fgn Ox, oxidised Fgn, CNBr, cyanogen bromide fragmented Fgn, and FDP-1 and FDP-2 are pooled samples from separate independent time courses of fibrin degradation products.</p

Model Parameters used in the model outlined in Fig 2 and simulated in Fig 3.

<p>Model Parameters used in the model outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.g002" target="_blank">Fig 2</a> and simulated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.g003" target="_blank">Fig 3</a>.</p

Kinetic parameters for clot lysis by tPA, rSK-M1GAS and rSK-H46A from data shown in Fig 5.

<p>Kinetic parameters for clot lysis by tPA, rSK-M1GAS and rSK-H46A from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.g005" target="_blank">Fig 5</a>.</p

Comparison of Pgn activation data and simulated data for rSK-M1GAS over a range of Pgn and Fgn concentrations.

<p>Panels A (experimental data) and C (simulated data) show fitted surface plots of rate of Pm generation plotted against Fgn and Pgn concentrations as shown, for 1.6 nM rSK-M1GAS. Panels B and D present the same results as a surface and contour plots giving rates of Pm production in pM/s against Pgn concentration (0–1.6 μM) and Fgn concentration (log scale for 0–30 μM). Panel E is an overlay of surfaces for the data and simulation shown in panels A and B. Experimental data using rSK-M1GAS at 1.6 (closed circles) and 0.4 nM (open squares) over a range of Pgn concentrations were fitted to the Michaelis-Menten equation to determine k_cat and K_M values, and calculate k_cat/ K_M at each Fgn concentration and this is shown in panel F. The solid line is for the same values calculated from simulated data using the same ranges of Pgn and Fgn (the lines overlap for 2 hypothetical rSK-M1GAS concentrations of 1.6 and 0.4 nM. R scripts and data files are provided in Supporting Information.</p